Bacillus anthracis, the causative agent of anthrax, is a spore-forming organism. The ability to prepare anthrax spores inexpensively and deliver them in an aerosol form, and the high mortality rate of inhalation anthrax, have made B. anthracis one of the most dreaded agents of biowarfare and bioterrorism. This bacterium encodes two toxins, lethal toxin (LF) and edema toxin (EF), which are collectively called "anthrax toxin" (AT). Lethal toxin is assembled from protective antigen (PA, 83 kDa) and lethal factor (LF, 90 kDa), and as its name implies, is primarily responsible for lethality from anthrax. Edema toxin is assembled from PA and Edema Factor (EF, 89 kDa), and has as its gross manifestation edema at the site of injection. Uptake of each of these toxins is critically dependent upon interaction of PA with its cellular receptor, which leads to receptor-mediated endocytosis followed by toxin translocation into the cytosol across the membrane of an acidic pH endosome. However, the precise understanding of how these toxins are taken up into cells, and the ability to intervene therapeutically in this process, has been severely impaired by not knowing the identity of the PA receptor. Therefore, the primary goal of the proposed research is to isolate and characterize the cellular receptor for PA. To this end, the applicant will use a genetic complementation approach that employs, as recipient cells, mutant CHO-K1 cell lines generated by the applicant and then subjected to a selection protocol to identify those lacking PA receptors. Once this receptor has been identified, the applicant will localize the PA binding domain, characterize the nature of its binding interaction with PA, and attempt to define the minimal cellular components required for AT assembly and translocation across membranes. The applicant will also determine how this receptor is taken up into cells after PA binding and oligomerization, by defining determinants of the receptor and cellular factors that are required for this process. Libraries of chemical compounds will be screened for effective inhibitors of the early steps of AT uptake and translocation. Collectively, these studies will significantly increase the knowledge of how anthrax toxin is taken up into cells and should identify effective chemical inhibitors of this process for future drug development.